1. Field of the Invention
The present invention relates generally to the fields of neurobiology and neurophysiology. More particularly, it concerns the methods and compositions for measuring the activity of neurotransmitter transporters. The invention also provides screening methods for identifying modulators of neurotransmitter transport.
2. Description of Related Art
Neurotransmitters mediate signal transduction in the nervous system and modulate the processing of responses to a variety of sensory and physiological stimuli. An important regulatory step in neurotransmission is the inactivation of a neurotransmitter following its release into the synaptic cleft. This is especially true for the biogenic amine and amino acid neurotransmitters. Inactivation of a neurotransmitter is typically mediated by uptake of the released neurotransmitter by neurotransmitter transporters that are located on the presynaptic neuron or in some cases on adjacent glial cells. Thus, neurotransmitter transporters are central to the processing of information in the nervous system and are associated with numerous neurological disorders.
For example, the neurotransmitter norepinephrine (also called noradrenalin) transduces signaling in the central nervous system that modulates attention, mood, arousal, learning, and memory (Aston-Jones et al., 1999; Coull et al., 1999; Skrebitsky and Chepkova, 1998; Hatfield and McGaugh, 1999). Norepinephrine (NE) transporters (NETs) attenuate neuronal signaling via rapid neurotransmitter clearance (Ressler and Nemeroff, 1999; Iversen et al., 1967; Axelrod and Kopin, 1969; Blakely et al., 1991). Norepinephrine transport is implicated in the pathology of major depression, post-traumatic stress disorder and attention deficit disorder (Ressler and Nemeroff, 1999; Southwick et al., 1999; Dow and Kline, 1997; Biederman and Spencer, 1999). Therapeutic agents that inhibit NET can elevate the concentration of norepinephrine in the brain and periphery (Axelrod and Kopin, 1969; Bonisch, 1984; Ramamoorthy et al., 1993; Galli et al., 1995; Corey et al., 1994; Fleckenstein et al., 1999). Noradrenergic signaling in the peripheral nervous system influences blood pressure and heart rate (Jones, 1991; Jacob et al., 1999; Hartzell, 1980), and NET inhibitors, such as cocaine and antidepressants, induce cardiac complications (Watanabe et al., 1981; Clarkson et al., 1993; Glassman et al., 1985).
Similarly other neurotransmitters such as epinephrine (E), dopamine (DA), serotonin (SE), and their respective transporters such as epinephrine transporters (ET), dopamine transporters (DAT), and the serotonin transporters (SERT), mediate diverse aspects of neuronal signaling and are involved in the pathology of numerous nervous system related disorders. Thus, neurotransmitter transporters are the targets of various therapeutic agents used in the treatment of neurological disorders including, depression, epilepsy, schizophrenia, Parkinson's disease, attention deficit disorders, eating and sleeping disorders as well as some neurodegenerative disorders. In some instances, treatment of these disorders is mediated by the use of pharmaceutical agents that are antagonists of a neurotransmitter transporter. Antagonists block uptake and prolong and/or enhance the action of the neurotransmitter. In other instances, treatment is mediated by use of pharmaceutical agents that are agonists of a neurotransmitter transporter. Agonists enhance uptake and rapidly clear the neurotransmitter, thereby terminating its actions. For example, imipramine, a blocker of SE and NE uptake, is used as an antidepressants; benztropine, an antagonist of dopamine uptake, temporarily alleviates the symptoms of Parkinson's disease; and blockers of γ-amino butyric acid (GABA) uptake are used in the treatment of epilepsy.
Despite the relevance of neurotransmitter transporters, the art is hindered by very limited methods that are used in studying neurotransmitter transporter functions such as kinetics, affinity, temporal and spatial aspects of transport, voltage dependence and other transport mechanics (Galli et al., 1995; Corey et al., 1994; DeFelice & Galli, 1998; Prasad and Amara, 2001). Methods used to study neurotransmitter transport typically involve the use of radiometric substrates to measure neurotransmitter accumulation. For example, 3H-labeled neurotransmitters are typically used to study transport of serotonin, epinephrine, norepinephrine, dopamine and the amino-acid transmitters (see for example U.S. Pat. No. 5,424,185; Bonisch 1984; Bonisch and Harder, 1986; Hadrich et al., 1999). Although radiolabel techniques offer high specificity these approaches have significant limitations such as poor time and spatial resolution. In addition, none of these methods have the intrinsic capability to distinguish substrate binding from transport in the same assay. For example, non-permeating radiolabeled molecules that bind neurotransporters can characterize binding and count transporters, and permeating radiolabeled molecules can characterize transport, however, because of the poor time resolution of radiometric assays it is not possible to study binding and transport during the same experiment. Furthermore, these methods are not applicable for studying transport function in single mammalian cells. Although electrophysiology and amperometry alleviate some of these constraints, electrophysiology although rapid (in the millisecond time resolution) has poor substrate selectivity, while amperometry has the reverse characteristics (DeFelice and Galli, 1998; Galli et al., 1998).
Several other studies involved the use of fluorescent analogs of neurotransmitters for the study of neurotransmitter transporters. For example, Hadrich and colleagues generated fluorescent NE and nisoxetine analogs to image neuroblastomas (Hadrich et al., 1999) and Bruns (1998) used a autofluorescent analog of serotonin (5-HT), 5,7-dihydrotryptamine to identify a serotonin uptake current in leech neurons, however, these fluorescent compounds were also unable to distinguish substrate binding from transport. Additionally, fluorescent substrates based on neurotransmitter structures have the capability of activating cell surface receptors for the neurotransmitter during a transporter assay and causing indirect effects on transport activity, lessening their utility. Thus, new methods for the analysis of neurotransmitter transport function are highly desirable.
The present inventors previously reported the fluorescent substrate 4-(4-dimethylaminostyrl)-N-methylpyridinium (ASP+) is transported by NET, DAT and SERT as well as methods to measure neurotransmitter transport mechanisms using ASP+ and fluorescence microscopy. U.S. Publication No. 20040115703. ASP+ was only minimally effective as a substrate for the serotonin transporter and exhibited transport by several other endogenous transporters that decrease signal to noise in transport assays, and also required the addition of fluorescence quenchers. Thus, additional substrates with distinct characteristics are needed to advance the understanding of neurotransmitter function.